Pseudomonas aeruginosa is the most predominant bacterial pathogen found in the lungs of cystic fibrosis (CF) patients, infecting 80% of them by the age of 18. Chronic P. aeruginosa infections are highly refractive even to aggressive antibiotic therapy, and almost always lead to rapid respiratory decline and early death among CF patients. Transition to chronic infection is invariably marked by the appearance of mucoid P. aeruginosa due to overproduction of a polysaccharide called alginate. The proposed research focuses on characterizing the complex regulatory cascade that leads to alginate production. The study has four specific aims: (1) Determining the hierarchy and targets of known players in the proteolytic cascade of P. aeruginosa; (2) Delineating the role of LptD/OstA, a protein involved in LPS biogenesis, in alginate production (3) Identifying novel genes by characterizing three suppressors of alginate production (sap) strains containing no mutations in known alg genes; and (4) Finding novel therapeutic strategies by using compounds that inhibit proteases involved in alginate production. The study will use a holistic approach by creating clean in-frame deletions and overexpression constructs of all known players; and investigating the functional order of action among different proteins using genetic epistasis and biochemical methods. The impact of the proposed research on public health is significant because it: (i) addresses a major cause of morbidity and mortality among CF patients; (ii) targets one of today's biggest public health threats: the development of bacterial resistance to current antibiotics; and (iii) moves the field forward by characterizing never-before-identified players in P. aeruginosa alginate regulatory cascade. The study is highly innovative because it: (a) takes a comprehensive approach to study the intricate alginate regulatory pathway; (b) uses a mucoid strain PDO300, isogenic to nonmucoid PAO1, to study alginate regulation; (c) explores novel players in alginate regulation; (d) delineates the role of previously unknown alginate players such as LptD/OstA; and (e) targets protease inhibitors as therapeutic agents that can impede chronic P. aeruginosa infections. Protease inhibitors have been successfully applied to treat viruses like HIV and Hepatitis C, but not against bacterial pathogens such as P. aeruginosa. The outcome of this proposal will lead to the development of new drugs and strategies that will benefit not only CF patients but others with bacterial infections that have developed resistance to commonly used antibiotics.